title: Specific visualization of nitric oxide in the vasculature with two-photon microscopy using a copper based fluorescent probe.
abstract: To study the role and (sub) cellular nitric oxide (NO) constitution in various disease processes, its direct and specific detection in living cells and tissues is a major requirement. Several methods are available to measure the oxidation products of NO, but the detection of NO itself has proved challenging. We visualized NO production using a NO-sensitive copper-based fluorescent probe (Cu 2FL2E) and two-photon laser scanning microscopy (TPLSM). Cu 2FL2E demonstrated high sensitivity and specificity for NO synthesis, combined with low cytotoxicity. Furthermore, Cu 2FL2E showed superior sensitivity over the conventionally used Griess assay. NO specificity of Cu 2FL2E was confirmed in vitro in human coronary arterial endothelial cells and porcine aortic endothelial cells using various triggers for NO production. Using TPLSM on ex vivo mounted murine carotid artery and aorta, the applicability of the probe to image NO production in both endothelial cells and smooth muscle cells was shown. NO-production and time course was detected for multiple stimuli such as flow, acetylcholine and hydrogen peroxide and its correlation with vasodilation was demonstrated. NO-specific fluorescence and vasodilation was abrogated in the presence of NO-synthesis blocker L-NAME. Finally, the influence of carotid precontraction and vasorelaxation validated the functional properties of vessels. Specific visualization of NO production in vessels with Cu 2FL2E-TPLSM provides a valid method for studying spatial-temporal synthesis of NO in vascular biology at an unprecedented level. This approach enables investigation of the pathways involved in the complex interplay between NO and vascular (dys) function.
authors.affiliation: Department of Genetics & Cell Biology-Molecular Biology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands ; Institute for Molecular Cardiovascular Research (IMCAR), RWTH University Aachen, Aachen, Germany.
authors.forename: Mitrajit
authors.initials: M
authors.lastname: Ghosh
authors.forename: Nynke M S
authors.initials: NM
authors.lastname: van den Akker
authors.forename: Karolina A P
authors.initials: KA
authors.lastname: Wijnands
authors.forename: Martijn
authors.initials: M
authors.lastname: Poeze
authors.forename: Christian
authors.initials: C
authors.lastname: Weber
authors.forename: Lindsey E
authors.initials: LE
authors.lastname: McQuade
authors.forename: Michael D
authors.initials: MD
authors.lastname: Pluth
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lippard
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Post
authors.forename: Daniel G M
authors.initials: DG
authors.lastname: Molin
authors.forename: Marc A M J
authors.initials: MA
authors.lastname: van Zandvoort
mesh_terms: D000818:Animals; D003331:Coronary Vessels / Q000166:cytology*; D042783:Endothelial Cells / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D016014:Linear Models; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015394:Molecular Structure; D019331:NG-Nitroarginine Methyl Ester; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D013552:Swine
chemical_list: D005456:Fluorescent Dyes; D009569:Nitric Oxide; D019331:NG-Nitroarginine Methyl Ester